Chroman-derived anti-androgens for treatment of androgen-mediated disorders

ABSTRACT

Methods for the prevention and/or alleviation of androgen-mediated disorders treatable by administering a chroman-derived anti-androgen compound are provided by the present invention. The invention further provides pharmaceutical and nutraceutical compositions containing chroman-derived anti-androgen compounds useful in the prevention and/or alleviation of androgen-mediated disorders, particularly prostate cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional application60/450,510, filed Feb. 27, 2003, incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This work was supported in part by grants from the Department ofDefense: DAMD17-98-1-8505, DAMD17-02-1-166 and DAMD17-99-1-9531. TheGovernment of the United States of America may have certain rights inthis invention.

FIELD OF THE INVENTION

This invention relates generally to chemical antagonists of the androgenreceptor. In particular, this invention is directed to chroman-derivedanti-androgens and methods of their use for preventing and/oralleviating androgen-mediated disorders such as prostate cancer.

BACKGROUND OF THE INVENTION

As a group, the male sex hormones are termed androgens. Among theandrogens, testosterone plays a central role in developing andmaintaining secondary male sexual characteristics, including: (1)enlargement of the male sex organs, prostate gland, seminal vesicles andbulbourethral glands; (2) increased growth of body hair, particularly onthe face and chest, but sometimes accompanied by decreased growth ofhair on the scalp; (3) enlargement of the larynx and thickening of thevocal cords; (4) thickening of the skin; (5) increased muscular growth;and (6) thickening and strengthening of the bones.

Testosterone is normally produced and secreted by interstitial cells ofthe testes under the influence of luteinizing hormone (LH). LH is agonadotropin secreted from the anterior lobe of the pituitary gland inresponse to yet another factor secreted from the hypothalamus, termedluteinizing hormone-release factor (LH-RF). The degree to which malesecondary characteristics develop is directly related to the amount oftestosterone secreted by the interstitial cells of the testes. Thisoverall amount of testosterone is regulated by a negative feedbacksystem involving the hypothalamus. As the concentration of testosteronein the blood increases, the hypothalamus senses the testosterone viaandrogen receptors and becomes inhibited, and its stimulation of theanterior pituitary gland by LH-RF is consequently decreased. As thepituitary's secretion of LH is reduced the amount of testosteronereleased by the interstitial cells of the testes is reduced also.However, as the blood level of testosterone drops, the hypothalamusbecomes less inhibited, and it once again stimulates the pituitary glandto release LH. The increasing secretion of LH causes the interstitialcells to release more testosterone, and its blood level rises.

As can be appreciated from the variety of secondary male sexualcharacteristics, the body possesses a plethora of sex hormone responsivetissues and organs. Unfortunately, many cancers types exhibitsusceptibility to sex hormone control mechanisms that regulate growth ofthe normal organ or tissue from which the neoplasm arose. On thepositive side, cancers originating in endocrine organs and the immunesystem are especially susceptible to medical therapies based on sexhormones, sex hormone antagonists, and/or hormone deprivation. In fact,the sex hormones and their antagonists represent useful agents for thetreatment of common cancers arising from the breast, prostate gland, anduterus.

In this regard, the role of traditional surgery in endocrine ablationhas diminished as chemical agents have been identified which can replacesurgical procedures. For example, surgical castration, also termedorchiectomy, useful in slowing or preventing the progression ofandrogen-mediated prostate cancer may be “chemically” achieved byadministering an anti-androgen in combination with a known LH-RFagonist. The anti-androgen/LH-RF agonist combination effectively lowersthe level of testosterone which, if left unchecked, increases the growthrate of testosterone-dependent prostatic neoplasias. RepresentativeLH-RF agonists include leuprolide or goserelin, described in U.S. Pat.Nos. 4,897,256 and 5,510,460, respectively. Useful anti-androgensinclude flutamide, bicalutamide, or nilutamide. Flutamide is anonsteroidal antagonist of the androgen receptor sold under thetradename Eulexin, as described in U.S. Pat. Nos. 3,995,060 and4,474,813. Bicalutamide is a nonsteroidal antagonist of the androgenreceptor sold under the tradename Casodex, as described in U.S. Pat. No.4,636,505. Nilutamide is also a nonsteroidal antagonist of the androgenreceptor and is sold under the tradename Nilandron, as described in U.S.Pat. No. 5,023,088.

Unfortunately, the hormonal therapies for prostatic cancer, whileoffering many patients a noninvasive option to drastic surgicalprocedures, are commonly accompanied by many complications or sideeffects. LH-RF agonists including leuprolide and goserelin act to lowertestosterone to post-castration levels but these agonists also result inimpotence and hot flashes. As well, anti-androgens targeting theandrogen receptor, including flutamide and bicalutamide, often causediarrhea, breast enlargement (a.k.a., gynecomastia), loss of libido, andnausea (Soloway, M. S., et al., Urology 47 (Suppl 1A): 33-37, 1996).There have also been case reports of toxic liver effects (Wysowski, D.D., et al., Annals of Internal Medicine 118(11): 860-864, 1993).

In part, the side effects observed in current chemical therapies are dueto the undesirable characteristic of current anti-androgen compounds tocross the blood brain barrier and affect androgen receptors of thecentral nervous system, apart from peripheral tissues. While androgenreceptors have been well studied in the hypothalamus and peripheraltissues, little is known about the actual molecular mechanisms thatresult in complications including, but not limited to, loss of libidoand nausea. Thus, the penetration of the blood brain barrier by currentagents is undesirable and improved agents targeting primarily peripheraltissues are extremely desirable.

Another undesirable effect of some of the current anti-androgenic agentsis their undesirable ability to exert partial agonist activity in someprostate cancer cells. For example, the anti-androgen flutamide has beenshown to stimulate, instead of inhibit, the growth of LNCaP humanprostate carcinoma cells in the laboratory setting (The Prostate 14:103-115 (1989)). This could potentially stimulate, instead of inhibit,the growth of prostate cancers in a subset of patients. Therefore, themost favorable anti-androgens should exhibit pure antagonist activity inregard to the androgen receptor, no matter their biological context(i.e., never act as androgen receptor agonists).

While anti-androgen compounds find use in cancer therapies, thesecompounds have also found utility in non-cancer-related hormonetherapies. For example, androgen-dependent hirsutism, manifest as excesshair in women, is currently treated with the anti-androgen flutamide.Unfortunately, many of the same side effects described above areexperienced by women treated with flutamide due to the general nature offlutamide's antagonist activity.

As can be readily appreciated, the quality of life afforded by currenthormone therapies, in particular therapies utilizing anti-androgens, isfar less than desirable. Therefore, there exists a need foranti-androgens that offer patients reduced complications while providingeffective regimens of hormone therapy. Anti-androgens exhibitingperipheral tissue-specific targeting would be extremely valuable inimproving the quality of hormone therapy available to those in needthereof.

SUMMARY OF THE INVENTION

The present invention is based on the inventor's pioneering discoverythat the chromanol-derived moiety of vitamin E possesses potentanti-androgenic activity in androgen-dependent cells. In particular, thecompound 2,2,5,7,8-pentamethyl-6-chromanol (PMCol) was identified by theinventors as demonstrating pure antagonist activity toward the androgenreceptor in prostate carcinoma cell lines. The anti-androgen activity ofchromanol-derived compounds was heretofore unknown. The variousembodiments of the invention described and claimed herein thusly provideadvantageous methods and compositions based on the inventors' unexpectedfindings.

In one embodiment, the invention is directed to a method for inhibitingthe growth of androgen-dependent tumor cells. The method includes thestep of administering to the tumor cells an effective amount of ananti-androgen compound according to Formula I:

-   -   wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₉ and R₁₀ are independently        a substituted or un-substituted C₁-C₃ alkyl group or H; and R₈        is an OH. The anti-androgen compound is water soluble and, in a        most preferred embodiment, the anti-androgen compound has the        structure of Formula II:

In another embodiment, the invention is a method of delaying theprogression of prostate cancer in a patient suffering from prostatecancer. The method includes the step of administering to the patient aneffective amount of anti-androgen compound according to Formula I. Theanti-androgen compound is water soluble and, in a most preferredembodiment, the anti-androgen compound has the structure of Formula II.

In another embodiment, the present invention is a method of preventingthe occurrence or recurrence of prostate cancer in a patient at riskthereof. The method includes the step of administering to the patient aneffective amount of anti-androgen compound according to Formula I. Theanti-androgen compound is water soluble and, in a most preferredembodiment, the anti-androgen compound has the structure of Formula II.

In one embodiment of the invention, a method for the treatment of anandrogen-mediated disorder remediable by contacting an androgen receptorwith an anti-androgen compound is provided. The method includes the stepof administering to a patient an effective amount of an anti-androgencompound having Formula I or its pharmaceutically acceptable salt. Inpreferred embodiments, the anti-androgen compound reversibly binds toand acts as antagonist of the androgen receptor. The anti-androgencompound is water soluble and, in a most preferred embodiment, theanti-androgen compound has the structure of Formula II.

According to the invention, the androgen-mediated disorder remediable bycontacting an androgen receptor with an anti-androgen compound accordingto Formula I may be, but is not limited to, hirsutism, acne, seborrhea,Alzheimer's disease, androgenic alopecia, hyperpilosity, benignprostatic hypertrophy, adenomas or neoplasias of the prostate, treatmentof benign or malignant tumor cells containing the androgen receptor,modulation of VEGF expression for use as antiangiogenic agents,osteoporosis, suppressing spermatogenesis, libido, cachexia,endometriosis, polycystic ovary syndrome, anorexia, androgen-relateddiseases and conditions, and male and female sexual dysfunction orinfertility. A preferred use of an anti-androgen compound describedherein is in the treatment or prevention of prostate cancer.

The present invention is also directed to pharmaceutical andnutraceutical compositions comprising an anti-androgen compound havingFormula I in combination with an acceptable carrier.

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structure of vitamin E (i.e., α-tocopherol) and relatedcompounds. A, α-tocopherol. B, 2,2,5,7,8-pentamethyl-6-chromanol(PMCol). C, 2,2,5,7,8-pentamethylchroman (PMC).

FIG. 2. PMCol competition analysis of R1881 binding in human prostatecarcinoma cells. A, A dose-response for the competition of PMCol, PMC,and bicalutamide for androgen receptor binding to ³H-R1881 wasdetermined in LNCaP cells. B, Competition for ³H-R1881 binding in LAPC4cells was determined for 30 μM PMCol and 1 μM bicalutamide. (*P<0.05;n=4.)

FIG. 3. Growth modulation of human prostate carcinoma cells by PMCol. A,Dose-response of DU145, LAPC4, and LNCaP cells grown in mediumcontaining 5% serum measured 4 d after PMCol treatment. Treatment with50 μM PMCol significantly reduced LNCaP prostate cell growth, whereas aconcentration of 80 μM PMCol was required to significantly decreasegrowth in the androgen-independent DU145 prostate cell line (*P<0.05).B, The PMCol dose-response of LNCaP cell growth was determined in cellsexposed to androgen-deficient conditions (i.e., using medium containingreduced androgen levels) with or without the addition of agrowth-stimulatory dose of 0.05 nM R1881 or a growth-inhibitory dose of1.0 nM R1881. (* significantly different than 0 μM PMCol-treated cells;P<0.05; n=6.)

FIG. 4. Shifts in the R1881-stimulated biphasic LNCaP growth responsewere determined after treatment with 30 μM PMCol, 30 μM PMC, or 1 μMbicalutamide for 4 d. The inhibition of growth response is readilyapparent at 0.3 nM R1881 exposure, where LNCaP growth from PMCol, PMC,and bicalutamide treatment was equivalent to the growth response incontrol cells produced by exposure to only 0.03 nM R1881.

FIG. 5. Analysis of PMCol effects on androgen-induced PSA secretion fromLNCaP cells. PSA secretion was determined 48 h after exposure to agrowth stimulatory dose of 0.05 nM R1881 or a growth inhibitory dose of1.0 nM R1881 in the presence of 30 μM PMC, 30 μM PMCol, or 1 μMbicalutamide. (*P<0.05 compared to 0.05 nM R1881 treated cells; **P<0.05compared to 1.0 nM R1881 treated cells; n=3.)

FIG. 6. Androgen-induced MMTV promoter activity in LNCaP (A) and LAPC4(B) cells after PMCol treatment. A, The effects of 25 μM PMCol, 50 μMPMCol, and 1 μM bicalutamide treatment for 24 h on MMTV promoteractivity induced by R1881 was assessed in LNCaP cells. B, LAPC4 cellsexposed to 30 μM PMCol effectively inhibited androgen-induced MMTVpromoter activity. (*P<0.05; n=4.)

FIG. 7. Immunoblot analysis of AR protein levels. AR protein levels werenot significantly altered in LNCaP cells exposed to 30 μM PMC, 30 μMPMCol, or 1 μM bicalutamide for 5 d compared to AR levels in vehiclecontrol exposed cells. LNCaP cells were grown in medium containing 5%serum to provide endogenous serum androgens, thus allowinganti-androgenic modulation of AR protein levels. Large arrow points toAR protein bands and the small arrow points to β-actin protein bands.

FIG. 8. Acute oral toxicity data for mice. A. This graph shows that nosignificant change in animal body mass occurred after administration ofa single, high-dose of PMCol compared to vehicle control at 48 hoursafter PMCol administration. B. No significant difference in body masschange was observed in comparing mice treated daily with PMCol orvehicle over 10 days. C. No gross changes in organs were observed foreither PMCol-treated or control mice as exemplified by data on livermass which was not significantly changed in mice receiving PMCol dailyfor 10 days.

DETAILED DESCRIPTION OF THE INVENTION I. In General

Before the present methods are described, it is understood that thisinvention is not limited to the particular methodology, protocols, celllines, and reagents described, as these may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and equivalents thereof knownto those skilled in the art, and so forth. As well, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference for the purpose of describing anddisclosing the chemicals, cell lines, vectors, animals, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

Abbreviations used herein include: AR, androgen receptor; αCEHC,α-carboxyethylhydroxychroman; CSS, charcoal-stripped serum; DMEM,Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MMTV/LTR,Mouse mammary tumor virus long terminal repeat; PBS, phosphate-bufferedsaline; PMC, 2,2,5,7,8-pentamethylchroman; PMCol,2,2,5,7,8-pentamethyl-6-chromanol; PSA, prostate-specific antigen;R1881, methyltrienolone.

II. The Invention

The present invention provides methods of utilizing newly identifiedanti-androgen compounds. These compounds define a new subclass ofcompounds useful for preventing or treating a wide variety ofandrogen-mediated disorders. Compounds useful in the present invention,in particular 2,2,5,7,8-pentamethyl-6-chromanol (PMCol), are derivedfrom the anti-oxidant moiety of vitamin E and have unexpectedanti-androgen activity as non-steroidal ligands of the androgenreceptor. Because of the chemical structure of PMCol and compoundsstructurally similar thereto, compounds useful in the present inventionexhibit significant solubility in water. Such compounds are particularlydesirable as improved anti-androgens as they will not readily cross theblood-brain barrier in amounts significant enough to evoke changes inphysiological parameters affected by the androgen receptors of braintissues residing behind the blood-brain barrier. Accordingly, thepresent methods provide therapeutic effects by antagonizing androgenreceptors in substantially only peripheral tissues and organs, incontrast to prior androgen receptor antagonists. In general, compoundsuseful in the present invention will possess a water solubility greaterthan vitamin E; vitamin E is practically insoluble in water but freelysoluble in acetone, ether or equivalent fat solvents. Furthermore, theanti-androgen compounds used according to the invention are pureantagonists and do not exhibit even partial agonist activity, as assayedin, for example, LNCaP human prostate carcinoma cells.

In one particular embodiment, the invention is directed to a method forinhibiting the growth of androgen-dependent tumor cells. This methodincludes the step of administering to the tumor cells an effectiveamount of an anti-androgen compound represented by the structure ofFormula I:

-   -   wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₉ and R₁₀ are independently        a substituted or un-substituted C₁-C₃ alkyl group or H; and R₈        is an OH. In a preferred embodiment, the above-described method        utilizes an anti-androgen compound having Formula II:

In Formula I, the substituent R is defined as an alkyl group, H or OH,unless otherwise indicated. An “alkyl” group refers to a saturatedaliphatic hydrocarbon. The alkyl group has 1-3 carbons, and may beun-substituted or substituted by one or more groups selected fromhalogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido,nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl.

A “hydroxy” group refers to an OH group. An “alkoxy” group refers to an—O-alkyl group wherein alkyl is as defined above. A “thio” group refersto an —SH group. A “thioalkyl” group refers to an —SR group wherein R isalkyl as defined above. An “amino” group refers to an —NH₂ group. An“alkylamino” group refers to an —NHR group wherein R is alkyl is asdefined above. A “dialkylamino” group refers to an —NRR′ group wherein Rand R′ are all as defined above. An “amido” group refers to an —CONH₂.An “alkylamido” group refers to an —CONHR group wherein R is alkyl is asdefined above. A “dialkylamido” group refers to an —CONRR′ group whereinR and R′ are alkyl as defined above. A “nitro” group refers to an NO₂group. A “carboxyl” group refers to a COOH group.

As contemplated herein, the present invention relates to methods ofutilizing an anti-androgen compound and/or its analog, derivative,isomer, metabolite, pharmaceutically acceptable salt, hydrate, N-oxide,or combinations thereof in the treatment or prevention of anandrogen-mediated disorder (e.g., prostate cancer). In one embodiment,the invention relates to the use of an analog of the anti-androgencompound. In another embodiment, the invention relates to the use of aderivative of the anti-androgen compound. In another embodiment, theinvention relates to the use of an isomer of the anti-androgen compound.In another embodiment, the invention relates to the use of a metaboliteof the anti-androgen compound. In another embodiment, the inventionrelates to the use of a pharmaceutically acceptable salt of theanti-androgen compound. In another embodiment, the invention relates tothe use of a hydrate of the anti-androgen compound. In anotherembodiment, the invention relates to the use of an N-oxide of theanti-androgen compound.

The anti-androgen compounds useful in the present invention arechroman-derived chemicals which are either known or obtainable throughpurification schemes and/or syntheses known to those of skill in theart. For example, a preferred embodiment utilizes the compound ofstructure II, PMCol, which is available from commercial sources such asAldrich (Milwaukee, Wis.). Furthermore, compounds structurally-relatedto PMCol, as described herein, may be derived through methodologiesdisclosed by, for example, Pope et al. in Free Radic. Biol. Med. 33:807-817 (2002) and Carey et al. in Advanced Organic Chemistry, Parts Aand B, Kluwer Academic/Plenum Publishers, 4^(th) Ed. (2001). Thesynthesis of αCEHC, a metabolite of vitamin E, is described fully byPope et al. and workers with skill in the art may modify this teachingusing techniques known in the field without undue experimentation toarrive at structurally-similar compounds useful in the presentinvention. Briefly, αCEHC is synthesized in a 2-step process. In thefirst step, gamma-methyl-gamma-vinylbutyrolactone (MVBL) is synthesizedusing a Grignard reaction with ethyl levulinate and vinyl magnesiumbromide in anhydrous ether. The MVBL intermediate will be purified byhigh-performance liquid chromatography (HPLC). In the second step, (+/−)αCEHC is synthesized by the condensation of trimethylhydroquinone withMVBL in the presence of a Lewis acid and purified using HPLC. αCEHCpurity is assessed using nuclear magnetic resonance spectroscopy andliquid-chromatography/mass spectrometry (LC/MS).

By structural comparison, α-CEHC and PMCol are closely related,differing only by the addition of a carboxyethyl group at the 2 positionof the chromanol ring. The structure of α-CEHC is set forth below asFormula III:

However, the presence of this carboxyethyl group will alter the chemicalproperties of PMCol with the carboxyl moiety increasing the chargecharacter of the chromanol compound. The carboxyl moiety therebyincreases the compound's water-solubility and thusly promotes improvedassociation of the compound with androgen receptor in the peripheraltissues. The importance of water solubility to chroman-derived compoundsuseful in the present invention was described above.

As defined herein, the term “isomer” includes, but is not limited tooptical isomers and analogs, structural isomers and analogs,conformational isomers and analogs, and the like. In one embodiment,this invention encompasses the use of different optical isomers of ananti-androgen compound of Formula I. It will be appreciated by thoseskilled in the art that the anti-androgen compounds useful in thepresent invention may contain at least one chiral center. Accordingly,the compounds used in the methods of the present invention may exist in,and be isolated in, optically-active or racemic forms. Some compoundsmay also exhibit polymorphism. It is to be understood that the presentinvention encompasses the use of any racemic, optically-active,polymorphic, or stereroisomeric form, or mixtures thereof, which formpossesses properties useful in the treatment of androgen-relatedconditions described and claimed herein. In one embodiment, theanti-androgen compounds are the pure (R)-isomers. In another embodiment,the anti-androgen compounds are the pure (S)-isomers. In anotherembodiment, the compounds are a mixture of the (R) and the (S) isomers.In another embodiment, the compounds are a racemic mixture comprising anequal amount of the (R) and the (S) isomers. It is well known in the arthow to prepare optically-active forms (for example, by resolution of theracemic form by recrystallization techniques, by synthesis fromoptically-active starting materials, by chiral synthesis, or bychromatographic separation using a chiral stationary phase).

The invention includes the use of pharmaceutically acceptable salts ofamino-substituted compounds with organic and inorganic acids, forexample, citric acid and hydrochloric acid. The invention also includesN-oxides of the amino substituents of the compounds described herein.Pharmaceutically acceptable salts can also he prepared from the phenoliccompounds by treatment with inorganic bases, for example, sodiumhydroxide. Also, esters of the phenolic compounds can be made withaliphatic and aromatic carboxylic acids, for example, acetic acid andbenzoic acid esters. As used herein, the term “pharmaceuticallyacceptable salt” refers to a compound formulated from a base compoundwhich achieves substantially the same pharmaceutical effect as the basecompound.

This invention further includes method utilizing derivatives of theanti-androgen compounds. The term “derivatives” includes but is notlimited to ether derivatives, acid derivatives, amide derivatives, esterderivatives and the like. In addition, this invention further includesmethods utilizing hydrates of the anti-androgen compounds. The term“hydrate” includes but is not limited to hemihydrate, monohydrate,dihydrate, trihydrate and the like.

This invention further includes methods of utilizing metabolites of theanti-androgen compounds. The term “metabolite” means any substanceproduced from another substance by metabolism or a metabolic process.

As used herein, receptors for extracellular signaling molecules arecollectively referred to as “receptors”. Many receptors aretransmembrane proteins on a cell surface where they contact or bind anextracellular signaling molecule (i.e., a ligand). In this manner, thereceptors initiate a cascade of intracellular signals that alter thebehavior of the cell. In contrast, in some cases, the receptors arelocated within the cell and the signaling ligand must first enter thecell by passive or active transport to activate the receptor.

Steroid hormones are one example of small molecules that diffusedirectly across the plasma membrane of target cells and bind tointracellular receptors. These receptors are structurally related andconstitute the intracellular receptor superfamily (or steroid-hormonereceptor superfamily). Steroid hormone receptors include progesteronereceptors, estrogen receptors, androgen receptors, glucocorticoidreceptors, and mineralocorticoid receptors. The present invention isparticularly directed to androgen receptors. An androgen receptor is anandrogen receptor of any species of, for example, a mammal. In oneembodiment, the androgen receptor is an androgen receptor of a human.

The invention is directed to methods utilizing anti-androgen compoundswhich are antagonist compounds. A receptor antagonist is a substancewhich contacts or interacts with receptors and inactivates them. Thus,an anti-androgen compound useful in the invention binds and inactivatessteroidal hormone receptors.

Assays to measure the anti-androgen activity of chroman-derivedcompounds, as described herein, are well known to a person skilled inthe art. For example, androgen receptor antagonistic activity can bedetermined by monitoring the ability of a candidate anti-androgencompound to inhibit the growth of androgen-dependent tissue, an exampleof such an assay being provided in the following Example section.

The compounds useful in the present invention bind either reversibly orirreversibly to an androgen receptor. In one embodiment, theanti-androgen compound binds reversibly to an androgen receptor. Inanother embodiment, the anti-androgen compound binds reversibly to anandrogen receptor of a mammal. In another embodiment, the anti-androgencompound binds reversibly to an androgen receptor of a human. Reversiblebinding of a compound to a receptor means that a compound can dissociatefrom the receptor after binding.

In another embodiment, the anti-androgen compound binds irreversibly toan androgen receptor. In one embodiment, the anti-androgen compoundbinds irreversibly to an androgen receptor of a mammal. In anotherembodiment, the anti-androgen compound binds irreversibly to an androgenreceptor of a human. Thus, in one embodiment, the compounds of thepresent invention may contain a functional group (e.g. affinity label)that allows alkylation of the androgen receptor (i.e. covalent bondformation). In this case, the compounds are alkylating agents which bindirreversibly to the receptor and, accordingly, cannot be displaced by asteroid, such as the endogenous ligands dihydroxy testosterone (DHT) andtestosterone. An “alkylating agent” is defined herein as an agent whichalkylates (forms a covalent bond) with a cellular component, such asDNA, RNA or protein. For example, in one embodiment, an alkylating groupis an isocyanate moiety, an electrophilic group which forms covalentbonds with nucleophilic groups (N, O, S etc.) in cellular components. Inanother embodiment, an alkylating group is an isothiocyanate moiety,another electrophilic group which forms covalent bonds with nucleophilicgroups (N, O, S etc.) in cellular components. In another embodiment, analkylating group is a haloalkyl (CH₂X wherein X is halogen), anelectrophilic group which forms covalent bonds with nucleophilic groupsin cellular components. In another embodiment, an alkylating group is ahaloalkyl-amido (NH COCH₂X wherein X is halogen), an electrophilic groupwhich forms covalent bonds with nucleophilic groups in cellularcomponents.

In certain embodiments, the present invention is a method for thetreatment of a condition remediable by contacting an androgen receptorwith an anti-androgen compound represented by the structure of FormulaI. Compounds according to Formula I, either alone or in a pharmaceuticalcomposition, are useful in treating a wide variety of such conditionsincluding, but not limited to, hirsutism, acne, seborrhea, Alzheimer'sdisease, androgenic alopecia, hyperpilosity, benign prostatichypertrophy, adenomas or neoplasias of the prostate, treatment of benignor malignant tumor cells containing the androgen receptor, modulation ofVEGF expression for use as antiangiogenic agents, osteoporosis,suppressing spermatogenesis, libido, cachexia, endometriosis, polycysticovary syndrome, anorexia, androgen-related diseases and conditions, maleand female sexual dysfunction and infertility.

In another embodiment, the invention is a method of delaying theprogression of prostate cancer in a patient suffering from prostatecancer. The method includes the step of administering to the patient aneffective amount of anti-androgen compound according to Formula I. Theanti-androgen compound is water soluble and, in a most preferredembodiment, the anti-androgen compound has the structure of Formula II.

In yet another embodiment, the present invention is a method ofpreventing the occurrence or recurrence of prostate cancer in a patientat risk thereof. The method includes the step of administering to thepatient an effective amount of anti-androgen compound according toFormula I. The anti-androgen compound is water soluble and, in a mostpreferred embodiment, the anti-androgen compound has the structure ofFormula II.

As defined herein, “contacting” means that the anti-androgen compoundused in the present invention is introduced into a sample containing thereceptor in a test tube, flask, tissue culture, chip, array, plate,microplate, capillary, or the like, and incubated at a temperature andtime sufficient to permit binding of the anti-androgen compound to thereceptor. Methods for contacting the samples with the anti-androgencompound or other specific binding components are known to those skilledin the art and may be selected depending on the type of assay protocolto be run. Incubation methods are also standard and are known to thoseskilled in the art.

In another embodiment, the term “contacting” means that theanti-androgen compound used in the present invention is introduced intoa patient receiving treatment, and the compound is allowed to come incontact with the androgen receptor in vivo.

As used herein, the term “treating” includes preventative as well asdisorder remittent treatment. As used herein, the terms “reducing”,“suppressing” and “inhibiting” have their commonly understood meaning oflessening or decreasing. As used herein, the term “progression” meansincreasing in scope or severity, advancing, growing or becoming worse.As used herein, the term “recurrence” means the return of a diseaseafter a remission.

As used herein, the term “administering” refers to bringing a patient,tissue, organ or cells in contact with an anti-androgen compoundaccording to Formula I. As used herein, administration can beaccomplished in vitro, i.e. in a test tube, or in vivo, i.e. in cells ortissues of living organisms, for example, humans. In certainembodiments, the present invention encompasses administering thecompounds useful in the present invention to a patient or subject. A“patient” or “subject”, used equivalently herein, refers to a mammal,preferably a human, that either: (1) has an androgen-dependent disorderremediable or treatable by administration of the anti-androgen accordingto Formula I; or (2) is susceptible to an androgen-dependent disorderthat is preventable by administering the anti-androgen according toFormula I.

In one embodiment, the methods of the present invention compriseadministering an anti-androgen compound as the sole active ingredient.However, also encompassed within the scope of the present invention aremethods for hormone therapy, for treating prostate cancer, for delayingthe progression of prostate cancer, for preventing the onset of prostatecancer, and for preventing and/or treating the recurrence of prostatecancer, which comprise administering the anti-androgen compounds incombination with one or more therapeutic agents. These agents include,but are not limited to: LH-RF analogs, other reversible/irreversibleanti-androgens, anti-estrogens, anti-cancer drugs, 5-alpha reductaseinhibitors, aromatase inhibitors, progestins, or agents acting throughother nuclear hormone receptors.

Thus, in one embodiment, the present invention provides methods ofadministering compositions and pharmaceutical compositions comprising ananti-androgen in combination with an LH-RF analog. In anotherembodiment, the present invention provides methods of administeringcompositions and pharmaceutical compositions comprising an anti-androgencompound, in combination with another reversible anti-androgen. Inanother embodiment, the present invention provides administeringcompositions and pharmaceutical compositions comprising an anti-androgencompound, in combination with an anti-estrogen. In another embodiment,the present invention provides administering compositions andpharmaceutical compositions comprising an anti-androgen compound, incombination with an anticancer drug. In another embodiment, the presentinvention provides administering compositions and pharmaceuticalcompositions comprising an anti-androgen compound, in combination with a5-alpha reductase inhibitor. In another embodiment, the presentinvention provides administering compositions and pharmaceuticalcompositions comprising an anti-androgen compound, in combination withan aromatase inhibitor. In another embodiment, the present inventionprovides administering compositions and pharmaceutical compositionscomprising an anti-androgen compound, in combination with a progestin.In another embodiment, the present invention provides administeringcompositions and pharmaceutical compositions comprising an anti-androgencompound, in combination with an agent acting through other nuclearhormone receptors.

As used herein, “pharmaceutical composition” means therapeuticallyeffective amounts of the anti-androgen compound together with suitablediluents, preservatives, solubilizers, emulsifiers, and adjuvants,collectively “pharmaceutically-acceptable carriers.” As used herein, theterms “effective amount” and “therapeutically effective amount” refer tothe quantity of active therapeutic agent sufficient to yield a desiredtherapeutic response without undue adverse side effects such astoxicity, irritation, or allergic response. The specific “effectiveamount” will, obviously, vary with such factors as the particularcondition being treated, the physical condition of the patient, the typeof animal being treated, the duration of the treatment, the nature ofconcurrent therapy (if any), and the specific formulations employed andthe structure of the compounds or its derivatives. In this case, anamount would be deemed therapeutically effective if it resulted in oneor more of the following: (a) the prevention of an androgen-mediateddisorder (e.g., prostate cancer); and (b) the reversal or stabilizationof an androgen-mediated disorder (e.g., prostate cancer). The optimumeffective amounts can be readily determined by one of ordinary skill inthe art using routine experimentation.

Pharmaceutical compositions are liquids or lyophilized or otherwisedried formulations and include diluents of various buffer content (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength, additives such asalbumin or gelatin to prevent absorption to surfaces, detergents (e.g.,Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents(e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzylalcohol, parabens), bulking substances or tonicity modifiers (e.g.,lactose, mannitol), covalent attachment of polymers such as polyethyleneglycol to the protein, complexation with metal ions, or incorporation ofthe material into or onto particulate preparations of polymericcompounds such as polylactic acid, polglycolic acid, hydrogels, etc, oronto liposomes, microemulsions, micelles, milamellar or multilamellarvesicles, erythrocyte ghosts, or spheroplasts. Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance. Controlled or sustained releasecompositions include formulation in lipophilic depots (e.g., fattyacids, waxes, oils).

Also encompassed by the invention are methods of administeringparticulate compositions coated with polymers (e.g., poloxamers orpoloxamines). Other embodiments of the compositions incorporateparticulate forms protective coatings, protease inhibitors or permeationenhancers for various routes of administration, including parenteral,pulmonary, nasal and oral. In one embodiment the pharmaceuticalcomposition is administered parenterally, paracancerally,transmucosally, tansdermally, intramuscularly, intravenously,intradermally, subcutaneously, intraperitonealy, intraventricularly,intracranially and intratumorally.

Further, as used herein “pharmaceutically acceptable carriers” are wellknown to those skilled in the art and include, but are not limited to,0.01-0.1M and preferably 0.05M phosphate buffer or 0.9% saline.Additionally, such pharmaceutically acceptable carriers may be aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia.

Parenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's and fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, collating agents, inertgases and the like.

Controlled or sustained release compositions administerable according tothe invention include formulation in lipophilic depots (e.g. fattyacids, waxes, oils). Also comprehended by the invention are particulatecompositions coated with polymers (e.g. poloxamers or poloxamines) andthe compound coupled to antibodies directed against tissue-specificreceptors, ligands or antigens or coupled to ligands of tissue-specificreceptors.

Other embodiments of the compositions administered according to theinvention incorporate particulate forms, protective coatings, proteaseinhibitors or permeation enhancers for various routes of administration,including parenteral, pulmonary, nasal and oral.

Compounds modified by the covalent attachment of water-soluble polymerssuch as polyethylene glycol, copolymers of polyethylene glycol andpolypropylene glycol, carboxymethyl cellulose, dextran, polyvinylalcohol, polyvinylpyrrolidone or polyproline are known to exhibitsubstantially longer half-lives in blood following intravenous injectionthan do the corresponding unmodified compounds (Abuchowski et al., 1981;Newmark et al., 1982; and Katre et al., 1987). Such modifications mayalso increase the compound's solubility in aqueous solution, eliminateaggregation, enhance the physical and chemical stability of thecompound, and greatly reduce the immunogenicity and reactivity of thecompound. As a result, the desired in vivo biological activity may beachieved by the administration of such polymer-compound abducts lessfrequently or in lower doses than with the unmodified compound.

In yet another method according to the invention, a pharmaceuticalcomposition can be delivered in a controlled release system. Forexample, the agent may be administered using intravenous infusion, animplantable osmotic pump, a transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump may be used (see Langer,supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989). In another embodiment, polymeric materials can be used. In yetanother embodiment, a controlled release system can be placed inproximity to the therapeutic target, i.e., the prostate, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984).Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990).

The pharmaceutical preparation can comprise the anti-androgen compoundalone, or can further include a pharmaceutically acceptable carrier, andcan be in solid or liquid form such as tablets, powders, capsules,pellets, solutions, suspensions, elixirs, emulsions, gels, creams, orsuppositories, including rectal and urethral suppositories.Pharmaceutically acceptable carriers include gums, starches, sugars,cellulosic materials, and mixtures thereof. The pharmaceuticalpreparation containing the anti-androgen compound can be administered toa patient by, for example, subcutaneous implantation of a pellet. In afurther embodiment, a pellet provides for controlled release ofanti-androgen compound over a period of time. The preparation can alsobe administered by intravenous, intraarterial, or intramuscularinjection of a liquid preparation oral administration of a liquid orsolid preparation, or by topical application. Administration can also beaccomplished by use of a rectal suppository or a urethral suppository.

The pharmaceutical preparations administerable by the invention can beprepared by known dissolving, mixing, granulating, or tablet-formingprocesses. For oral administration, the anti-androgens or theirphysiologically tolerated derivatives such as salts, esters, N-oxides,and the like are mixed with additives customary for this purpose, suchas vehicles, stabilizers, or inert diluents, and converted by customarymethods into suitable forms for administration, such as tablets, coatedtablets, hard or soft gelatin capsules, aqueous, alcoholic or oilysolutions. Examples of suitable inert vehicles are conventional tabletbases such as lactose, sucrose, or cornstarch in combination withbinders such as acacia, cornstarch, gelatin, with disintegrating agentssuch as cornstarch, potato starch, alginic acid, or with a lubricantsuch as stearic acid or magnesium stearate.

Examples of suitable oily vehicles or solvents are vegetable or animaloils such as sunflower oil or fish-liver oil. Preparations can beeffected both as dry and as wet granules. For parenteral administration(subcutaneous, intravenous, intraarterial, or intramuscular injection),the anti-androgen compounds or their physiologically toleratedderivatives such as salts, esters, N-oxides, and the like are convertedinto a solution, suspension, or expulsion, if desired with thesubstances customary and suitable for this purpose, for example,solubilizers or other auxiliaries. Examples are sterile liquids such aswater and oils, with or without the addition of a surfactant and otherpharmaceutically acceptable adjuvants. Illustrative oils are those ofpetroleum, animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil, or mineral oil. In general, water, saline, aqueousdextrose and related sugar solutions, and glycols such as propyleneglycols or polyethylene glycol are preferred liquid carriers,particularly for injectable solutions.

The preparation of pharmaceutical compositions which contain an activecomponent is well understood in the art. Such compositions may beprepared as aerosols delivered to the nasopharynx or as injectables,either as liquid solutions or suspensions; however, solid forms suitablefor solution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like or any combination thereof.

In addition, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentswhich enhance the effectiveness of the active ingredient.

An active component can be formulated into the composition asneutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts, which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric, mandelic, and thelike. Salts formed from the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

For topical administration to body surfaces using, for example, creams,gels, drops, and the like, the anti-androgen compounds or theirphysiologically tolerated derivatives such as salts, esters, N-oxides,and the like are prepared and applied as solutions, suspensions, oremulsions in a physiologically acceptable diluent with or without apharmaceutical carrier.

In another method according to the invention, the active compound can bedelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,N.Y., pp. 353-365 (1989); Lopez-Berestein ibid., pp. 317-327; seegenerally ibid).

For use in medicine, the salts of the anti-androgen compound may bepharmaceutically acceptable salts. Other salts may, however, be usefulin the preparation of the compounds according to the invention or oftheir pharmaceutically acceptable salts. Suitable pharmaceuticallyacceptable salts of the compounds include acid addition salts which may,for example, be formed by mixing a solution of the compound according tothe invention with a solution of a pharmaceutically acceptable acid suchas hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaricacid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalicacid, citric acid, tartaric acid, carbonic acid or phosphoric acid.

In addition, the anti-androgen compounds described herein may beprovided in the form of nutraceutical compositions where theanti-androgen compound prevents the onset of or reduces or stabilizesvarious deleterious androgen-related disorders, e.g., prostate cancer.The term “nutraceutical,” or “nutraceutical composition”, for thepurposes of this specification, refers to a food item, or a part of afood item, that offers medical health benefits, including preventionand/or treatment of disease. A nutraceutical composition according tothe present invention may contain only an anti-androgen compoundaccording to the present invention as an active ingredient or,alternatively, may further comprise, in admixture with the aforesaidanti-androgen compound, dietary supplements including vitamins,co-enzymes, minerals, herbs, amino acids and the like which supplementthe diet by increasing the total intake of that substance.

Therefore, the present invention provides methods of providingnutraceutical benefits to a patient comprising the step of administeringto the patient a nutraceutical composition containing a compound havingFormula I or a pharmaceutically acceptable salt thereof. Suchcompositions generally include a “nutraceutically-acceptable carrier”which, as referred to herein, is any carrier suitable for oral deliveryincluding, but not limited to, the aforementionedpharmaceutically-acceptable carriers. In certain embodiments,nutraceutical compositions according to the invention comprise dietarysupplements which, defined on a functional basis, include immuneboosting agents, anti-inflammatory agents, anti-oxidant agents, ormixtures thereof.

The immune boosters and/or anti-viral agents are useful for acceleratingwound-healing and improved immune function; and they include extractsfrom the coneflowers, or herbs of the genus Echinacea, extracts fromherbs of the genus Sambuca, and Goldenseal extracts. Herbs of the genusAstragalus are also effective immune boosters in either their natural orprocessed forms. Astragalus stimulates development into of stem cells inthe marrow and lymph tissue active immune cells. Zinc and its bioactivesalts, such as zinc gluconate and zinc acetate, also act as immuneboosters in the treatment of the common cold.

Antioxidants include the natural, sulfur-containing amino acid allicin,which acts to increase the level of antioxidant enzymes in the blood.Herbs or herbal extracts, such as garlic, which contain allicin are alsoeffective antioxidants. The catechins, and the extracts of herbs such asgreen tea containing catechins, are also effective antioxidants.Extracts of the genus Astragalus also show antioxidant activity. Thebioflavonoids, such as quercetin, hesperidin, rutin, and mixturesthereof, are also effective as antioxidants. The primary beneficial roleof the bioflavonoids may be in protecting vitamin C from oxidation inthe body. This makes more vitamin C, or ascorbic acid, available for useby the body.

Bioflavonoids such as quercetin are also effective anti-inflammatoryagents, and may be used as such in the inventive compositions.Anti-inflammatory herbal supplements and anti-inflammatory compoundsderived from plants or herbs may also be used as anti-inflammatoryagents in the inventive composition. These include bromolain, aproteolytic enzyme found in pineapple; teas and extracts of stingingnettle; turmeric, extracts of turmeric, or curcumin, a yellow pigmentisolated from turmeric.

Another supplement which may be used in the present invention is ginger,derived from herbs of the genus Zingiber. This has been found to possesscardiotonic activity due to compounds such as gingerol and the relatedcompound shogaol as well as providing benefits in the treatment ofdizziness, and vestibular disorders. Ginger is also effective in thetreatment of nausea and other stomach disorders.

Supplements which assist in rebuilding soft tissue structures,particularly in rebuilding cartilage, are useful in compositions fortreating the pain of arthritis and other joint disorders. Glucosamine,glucosamine sulfate, chondroitin, and chondroitin sulfate areparticularly useful for this purpose. Chondroitin may be derived from avariety of sources, such as Elk Velvet Antler. Marine lipid complexes,omega 3 fatty acid complexes, and fish oil are also known to be usefulin treating pain associated with arthritis.

Supplements useful in treating migraine headaches include feverfew andGingko biloba. The main active ingredient in feverfew is thesesquiterpene lactone parthenolide, which inhibits the secretion ofprostaglandins which in turn cause pain through vasospastic activity inthe blood vessels. Feverfew also exhibits anti-inflammatory properties.Fish oil, owing to its platelet-stabilizing and antivasospastic actions,may also be useful in treating migraine headaches. The herb Gingkobiloba also assists in treatment of migraines by stabilizing arteriesand improving blood circulation.

Although some of the supplements listed above have been described as totheir pharmacological effects, other supplements may also be utilized inthe present invention and their effects are well documented in thescientific literature.

The following Examples are offered by way of illustration and not by wayof limitation.

III. Examples Example 1

Androgen antagonist activity by the antioxidant moiety of vitamin E,2,2,5,7,8-pentamethyl-6-chromanol, in human prostate carcinoma cells.

A. Summary

The present inventors have shown that the antioxidant moiety of vitaminE, 2,2,5,7,8-pentamethyl-6-chromanol (PMCol), has anti-androgen activityin prostate carcinoma cells. In the presence of PMCol, theandrogen-stimulated biphasic growth curve of LNCaP human prostatecarcinoma cells was shifted to the right. The PMCol-induced growth shiftwas similar to that produced by treatment with the pure anti-androgenbicalutamide (i.e., Casodex), indicative of androgen receptor antagonistactivity. The concentration of PMCol used was below the concentrationrequired to affect cell growth or viability in the absence of androgen.Using an androgen receptor binding competition assay, PMCol was found tobe a potent anti-androgen in both LNCaP and LAPC4 cells, with an IC₅₀ ofapproximately 10 μM against 1 nM R1881 (a stable, synthetic androgen).Prostate-specific antigen release from LNCaP cells produced by androgenexposure with either 0.05 or 1.0 nM R1881 was inhibited 100% and 80%,respectively, by 30 μM PMCol. Also, PMCol inhibited androgen-inducedpromoter activation in both LNCaP and LAPC4 cells. However, PMCol didnot affect androgen receptor protein levels, suggesting that theinhibitory effects of PMCol on androgenic pathways were not due todecreased expression of the androgen receptor. Therefore, growthmodulation by the antioxidant moiety of vitamin E in androgen-sensitiveprostate carcinoma cells is due, at least in part, to its potentanti-androgenic activity.

B. Background

The activity of androgens is tissue-specific and mediated through theandrogen receptor (AR). The disruption of androgens and AR activityalters the regulation of androgen-sensitive tissues, such as theprostate gland (1). In the prostate, androgens have a central role innormal glandular development and function (2). However, androgens arealso necessary for the development of prostate cancer. The role ofandrogens in prostate cancer development is emphasized by theobservation that eunuchs and men that have a mutation in 5α-reductasetype II, an enzyme that converts testosterone to the more potentdihydrotestosterone, do not develop prostate cancer (3). The incidenceof prostate cancer has continued to rise for the last two decades,currently affecting over 200,000 men in the United States each year (4).Agents that permit the necessary actions of androgen for normal tissuefunction while reducing the role of androgens in the pathogenesis ofandrogen-sensitive tissues may serve as a useful means of reducingprostate cancer development. Recently, several agents have been reportedto prevent prostate cancer development, such as selenium, lycopene, andvitamin E (5). Due to the biochemical nature of these agents they arebelieved to act primarily through antioxidant-related pathways. However,the scope of their biological activity has not been extensivelyinvestigated.

Vitamin E is a family of naturally occurring dietary factors, which wereoriginally identified as necessary for reproduction (6). α-tocopherol,the most potent form of vitamin E, has two main components; asixteen-carbon phytyl chain and a chromanol moiety with four methylgroup substitutions (7). Biologically, α-tocopherol is thought to actprimarily as an antioxidant, reducing oxidative damage to lipids. Thechromanol moiety of α-tocopherol is responsible for its antioxidantactivity, whereas the phytyl chain increases the lipophilicity ofα-tocopherol and contributes to its tissue and subcellular distribution(8). Cell culture studies using α-tocopherol are difficult to performdue to its limited water solubility. However, the antioxidant chromanolmoiety of α-tocopherol, PMCol, which does not possess a phytyl chain, issufficiently water soluble to permit studies in cell culture.α-tocopherol and PMCol are shown in FIG. 1 along with PMC.

Most human prostate carcinoma cell lines are androgen independent. TheLNCaP human prostate carcinoma cell line is one of the few cell lines toshow demonstrable responses to androgen exposure (9). Interestingly,LNCaP cells produce a biphasic growth response to androgen exposure withgrowth stimulation occurring at lower doses and growth inhibitionoccurring in the absence of androgen or in the presence of high androgenlevels (9, 10). In addition, a number of androgen-sensitive responsesare induced in LNCaP cells. For example, LNCaP cells produce adose-dependent increase in PSA expression on androgen exposure (11, 12).Also, androgen-sensitive promoters, such as the MMTV promoter, areactivated by androgen in LNCaP cells (13). The exquisite sensitivity ofLNCaP cells to androgenic stimulation may be due to a mutation in theligand-binding domain of the androgen receptor (14). To date, the LNCaPprostate cell line has been the most extensively characterized prostatecell line for examining the effects of androgens. More recently, theLAPC-4 cell line has been introduced as another androgen-sensitive humanprostate carcinoma cell line that expresses a normal AR (15). However,the response of LAPC-4 cells to androgens is not as pronounced asobserved in LNCaP cells. Collectively, the LNCaP and LAPC4 humanprostate carcinoma cell lines provide valuable models for investigatingandrogen-regulated cellular pathways.

Previous studies have focused primarily on the inhibition of prostatecell growth by vitamin E treatment, which may occur through effects oncell cycle regulators (16, 17, 18). Apoptotic responses induced byvitamin E treatment have also been observed in LNCaP cells (19, 20).Interestingly, vitamin E-induced apoptotic responses were enhanced bycoadministration of androgen (19). Zhang et al (21) reported thatvitamin E succinate reduces the levels of the AR in LNCaP cells, withresultant inhibition of androgen-mediated responses. However, the directactions of vitamin E and related compounds on androgen receptor activityin prostate cells have not been extensively examined. In the studydescribed below, the androgen receptor antagonist activity andmodulation of androgen-sensitive pathways by the vitamin E derivative,PMCol, were investigated by the present inventors in human prostatecarcinoma cells.

C. Materials and Methods

Chemicals. PMCol and PMC were obtained from Aldrich (Milwaukee, Wis.).The chemical structures of α-tocopherol, PMCol, and PMC are shown inFIG. 1. Bicalutamide (Casodex) was kindly provided by AstraZenecaPharmaceuticals (Wilmington, Del.). R1881 and ³H-R1881 (87 Ci/mmol) wereobtained from Perkin Elmer/NEN Life Science Products (Boston, Mass.).All other chemicals used in these studies were acquired from SigmaChemical Company (Saint Louis, Mo.).

Cell culture. LNCaP cells were acquired from American Type CultureCollection (Manassas, Va.) and LAPC4 cells were kindly provided by Dr.Robert Reiter (University of California—Los Angeles) and maintained inDMEM containing 5% heat-inactivated fetal calf serum (Sigma) withstreptomycin-penicillin antibiotics (designated DMEM/FBS) in a 5% CO₂incubator at 37° C. For experiments evaluating androgenic responses,cells were cultured in phenol red-free DMEM (Gibco/BRL, Carlsbad,Calif.) containing 4% charcoal-stripped fetal calf serum and 1%unstripped fetal calf serum (designated DMEM/CSS).

Androgen receptor binding competition assay. An androgen receptorbinding competition assay was performed as previously described (22).LNCaP or LAPC4 prostate carcinoma cells were plated in 12-well tissueculture dishes (Costar, Corning, N.Y.) at 3.0×10⁵ cells per well inphenol-red free DMEM/CSS 3 d prior to analysis. For competitor analysis,DMEM/CSS was removed by aspiration and replaced with 1 mL of phenol-redfree DMEM containing 1 nM ³H-R1881, 1 μM triamcinolone acetonide, andcompetitor at the specified concentrations for 2 h at 37° C. in a 5% CO₂incubator. After incubation, competitor solution was aspirated and cellswere removed from the plate by trypsinization and placed in 12×75 mmpolystyrene tubes. Cells were washed twice with 1 mL phenol red-freeDMEM and placed in 8.0 mL of ScintiVerse II Scintillation Cocktail(Fisher Scientific, Pittsburgh, Pa.) for determination of radioactivity(i.e., dpm) using a Beckman LS 6000TA Liquid Scintillation System(Beckman Instruments Inc., Fullerton, Calif.).

Cell growth and viability analyses. Five thousand LNCaP or LAPC4 cellswere plated in each well of 96-well plates (Costar) in 100 μl ofDMEM/CSS. Two to 3 d after plating, cells were treated by adding 100 μlof DMEM/CSS containing 2× the concentration of the specified treatmentto each well. Four d after treatment, the relative cell number wasestimated by the determining DNA concentration of each well using aHoechst-based fluorescence DNA assay, as previously described (23).Growth analysis with DU145 cells was performed similar to those withLNCaP and LAPC4 cells except DU145 cells were initially seeded at 500cells per well. Cell viability was determined by trypan blue exclusionand quantified by light microscopic analysis using a hemacytometer.

Determination of secreted PSA levels. LNCaP cells were cultured in 96well plates (Costar) at 5,000 cells per well in DMEM/CSS 1 d beforetreatment. Forty-eight h after treatment, PSA levels in cell culturemedia were determined using the Tandem-MP PSA kit (Beckman Coulter,Inc.) according to manufacturer's instructions. PSA levels werenormalized to DNA levels as determined using a Hoechst-basedfluorescence DNA assay (23).

Androgen-stimulated promoter reporter assay analysis. LNCaP and LAPC4prostate carcinoma cell lines were cultured in 12-well cell cultureplates (Costar) in DMEM/CSS 2 to 3 d before transfection.Androgen-induced trancriptional activation was determined using areporter construct with an MMTV promoter that regulates the expressionof luciferase (24). LNCaP and LAPC4 cells were transfected with theMMTV/luciferase plasmid using the Effectene Transfection Reagent (QiagenInc., Valencia, Calif.), according to the manufacturer's instructions.Twenty-four h after transfection, cells were treated with R1881 with orwithout test reagents at the specified concentrations. Cell extractswere acquired 24 to 48 h after treatment by removing medium, washing 1×with PBS, and obtaining extract with 200 μL of 1× Reporter Lysis Buffer(Promega, Madison, Wis.). Luciferase activity was determined aspreviously described (24).

Immunoblot analysis of AR protein levels. LNCaP cells were plated at adensity of 1×10⁶ cells per 100 mm cell culture plate in 10 ml ofDMEM/FBS and maintained in incubators at 37° C. in 5% CO₂. After 5 d oftreatment with vehicle, 30 μM PMC, 30 μM PMCol, or 1.0 μM bicalutamide,cells were washed in cold 1×PBS and lysed in a buffer containing 1.0%Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.1mg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 10μg/ml aprotinin in 1×PBS. Total protein (10 μg) from cell extracts wereelectrophoresed on 7.5% SDS-polyacrylamide gels and transferred toImmobilon-P membranes (Millipore Corp., Bedford, Mass.) using a GENIEwet transfer system (Idea Scientific, Minneapolis, Minn.). Membraneswere blocked in Tris-buffered saline containing 5% nonfat dry milk atand then incubated with mouse anti-AR (441) monoclonal antibody (SantaCruz Biotechnology, Santa Cruz, Calif.) and mouse anti-actin antibody(A5441; Sigma). Membranes were then incubated with a secondaryhorseradish peroxidase-conjugated anti-mouse antibody (AmershamPharmacia Biotech, Piscataway, N.J.) and analyzed using the EnhancedChemiluminescence Plus reagent (Amersham Pharmacia Biotech).Autoradiograms were prepared by exposing the blots to BioMax Light X-rayfilm (Eastman Kodak Co., Rochester, N.Y.) and developed using a CURIX 60CP Processor (Agfa, Ridgefield Park, N.J.).

Statistical analysis. Significant differences in values between groupswere assessed using a two-sided Student's T-test. P values less than0.05 were used to signify statistical significance.

D. Results

PMCol Inhibits Androgen Binding in Prostate Cancer Cells. AR competitionwas determined using ³H-R1881 in the androgen-sensitive LNCaP cell line,which expresses a functional mutant AR (25), and the LAPC4 cell line,which express a normal human AR (15). Increasing concentrations of theAR antagonist bicalutamide were found to progressively inhibit R1881binding (FIG. 2A), with an estimated IC₅₀ of 0.7 μM in LNCaP cells.PMCol was found to be approximately 10-fold less potent at competing for³H-R1881 than bicalutamide in LNCaP cells, with an estimated IC₅₀ of 7.2μM (FIG. 2A). Repeated studies of PMCol competition for ³H-R1881 bindinggave IC₅₀ values ranging from 5 to 15 μM (data not shown). In contrast,PMC, in which the 6-hydroxyl of PMCol is absent, had lessanti-androgenic activity than PMCol (FIG. 2A) and significantly reducedcell viability at a concentration of 100 μM within 2 h of treatment(data not shown). Based on the R1881 competition results in LNCaP cells(FIG. 2A), a dose of 30 μM PMC and PMCol was used in most of thesestudies, allowing an effective comparison of the anti-androgenicactivity between PMC and PMCol. In LAPC4 cells, treatment with 30 μMPMCol produced a 75% decrease in ³H-R1881 binding and treatment with 1μM bicalutamide produced a 62% decrease in ³H-R1881 binding (FIG. 2B).

Modulation of prostate carcinoma cell growth and viability by PMCol.Changes in growth of the androgen-independent DU145 prostate carcinomacell line and the androgen-sensitive LNCaP and LAPC4 prostate cell lineswere assessed at concentrations of PMCol ranging from 10 to 100 μM (FIG.3A). Concentrations of 50 μM, 60 μM, and 80 μM or more PMCol wererequired to significantly reduce cell growth in LNCaP, LAPC4, and DU145cells, respectively (FIG. 3A). LNCaP cells produce a biphasic growthresponse to androgen exposure (9). Modulation of LNCaP cell growth byPMCol treatment was examined over 4 d. PMCol had no growth modulatoryactivity in vehicle-control treated LNCaP cells grown inandrogen-deficient media (i.e., PMCol did not have AR agonist activity)at concentrations ranging from 10 μM to 30 μM PMCol (FIG. 3B). However,LNCaP cell growth was decreased at concentrations equal to or higherthan 40 μM PMCol (FIG. 3B) and PMCol concentrations of 100 μM or greaterproduced significant cell death at 48 and 96 h (Table I). Stimulation ofLNCaP growth by exposure to 0.1 nM R1881 was significantly inhibited bytreatment with concentrations of 10 μM or more PMCol (FIG. 3B). However,a significant stimulation in LNCaP cell growth was observed in thepresence of a normally growth inhibitory concentration of 1.0 nM R1881with treatment of 10 μM to 30 μM PMCol (FIG. 3B). The R1881-stimulatedgrowth curve of LNCaP cells was shifted to the right in the presence of30 μM PMCol, similar to that produced by treatment with 1 μMbicalutamide (FIG. 4). A more modest, but significant, shift to theright in the androgen-induced LNCaP growth curve was observed bytreatment with 30 μM PMC (FIG. 4).

TABLE 1 Time- and dose-dependent changes in LNCaP cell viability afterPMCol treatment % Cell Viability^(a) (SD) [PMCol] (μM) Time (h) 0 25 5075 100 250 48 92.3 (4.7) 90.0 (2.8) 88.0 (3.4) 80.0 (12.5) 71.0 (87)^(b)11.0 (8.6)^(b) 96 88.0 (2.5) 87.0 (4.8) 85.0 (4.6) 87.0 (4.0)  21.0(33)^(b)  2.0 (1.8)^(b) ^(a)Determined by trypan blue exclusion analysisand quantified using a hemacytometer. ^(b)Significantly differentcompared to 0 μM PMCol (P < 0.05; n = 4).

Inhibition of PSA secretion by PMCol in LNCaP cells. PSA secretion byLNCaP cells is stimulated by androgen exposure in a dose-dependentmanner (12). The R1881-stimulated production of PSA from LNCaP cells wasmeasured after PMCol treatment for 48 h. PSA release from LNCaP cellswas not affected by treatment with 30 μM PMCol alone (FIG. 5). However,PSA levels were increased 3.1-fold after exposure to a growthstimulatory dose of 0.05 nM R1881, which was completely inhibited bytreatment with 30 μM PMCol (FIG. 5). Exposure of LNCaP cells to 1.0 nMR1881 produced a 12-fold increase in PSA levels by 48 h, which wasdecreased 20%, 81%, and 43% by treatment with 30 μM PMC, 30 μM PMCol, or1 μM bicalutamide, respectively (FIG. 5).

Inhibition of androgen-stimulated transcriptional activation by PMCol.Studies on androgen-regulated transcriptional activation were performedin LNCaP and LAPC4 cells transiently transfected with a reporter vectorthat uses the androgen-sensitive MMTV/LTR to drive expression of aluciferase reporter gene. In LNCaP cells, PMCol treatment alone had noeffect on MMTV promoter activity, whereas luciferase expression wasincreased 54-fold after exposure to 1.0 nM R1881 for 24 h (FIG. 6A).Luciferase expression induced by exposure to 1.0 nM R1881 in LNCaP cellsfor 24 h was decreased 50% and 70% by treatment with 25 μM and 50 μMPMCol, respectively (FIG. 6A). Similarly, LAPC4 cells exposed to 1.0 nMR1881 produced a 20-fold increase in MMTV/LTR driven luciferaseexpression that was decreased 60% by treatment with 30 μM PMCol after 24h (FIG. 6B). In both LNCaP and LAPC4 cells, treatment with 1 μMbicalutamide decreased 1.0 nM R1881-stimulated luciferase expressionapproximately 50% (FIGS. 6A and 6B).

Androgen receptor protein levels in PMCol exposed LNCaP cells. Previousstudies in LNCaP cells have reported that AR levels are decreased aftertreatment with vitamin E analogs, which may account for the reducedsensitivity of these cells to androgen exposure (21). However, in thecurrent study, LNCaP cells treated with 30 μM PMC, 30 μM PMCol, or 1 μMbicalutamide for 5 d did not result in altered AR protein levels (FIG.7).

E. Discussion

In the current study, the inventors examined the effects of an agenttraditionally considered as an antioxidant on prostate carcinoma cells.Epidemiological studies provide intriguing evidence that antioxidantdietary factors such as β-lycopene and vitamin E may help preventprostate cancer development (5). Although these agents have beenclassified as antioxidants, the mechanism by which they may contributeto prostate cancer prevention has not been firmly established. Androgensare known to have an essential role in prostate cancer development (3).Modulation of androgen activity may provide a means of prostate cancerprevention (26). Here, the inventors have determined the antioxidantmoiety of vitamin E, PMCol, to be a potent anti-androgen inandrogen-sensitive human prostate carcinoma cells.

The LNCaP human prostate carcinoma cell line is one of the few prostatecell lines that show demonstrable physiologic changes resulting fromandrogen exposure, such as growth modulation (9). Therefore, the LNCaPcell line has proven valuable in identifying agents that alterandrogen-stimulated cell growth. In the current study, PMCol shifted theandrogen-mediated growth curve in LNCaP cells such that higher androgenconcentrations were necessary to produce the biphasic growth responsetypically observed in LNCaP cells. The LNCaP growth shift with PMColtreatment was sufficient to produce growth stimulation in the presenceof 1.0 nM R1881, a concentration of R1881 that typically inhibits LNCaPproliferation (10). The shift in LNCaP growth pattern observed withPMCol treatment was similar to that observed in LNCaP cells aftertreatment with the pure anti-androgen bicalutamide. Also, the IC₅₀ ofPMCol observed in an androgen competition analysis for R1881 binding inLNCaP cells is in agreement with the dose-response shift inandrogen-mediated growth of LNCaP cells after PMCol treatment. Together,these results suggest that the shift observed in the androgen-mediatedgrowth of LNCaP cells was due to the anti-androgenic activity of PMCol.

Although LNCaP cells have proven to be useful in evaluatingandrogen-responsive pathways, the use of LNCaP cells to assessanti-androgenic activity can be inaccurate since LNCaP cells harbor amutant AR (25). The AR receptor in LNCaP cells, which althoughfunctional, has been reported to have altered ligand binding affinity(14) and is stimulated by some agents that are antagonists for thewild-type AR (22). Therefore, in this study, competition for AR bindingby PMCol was also assessed in the LAPC4 human prostate carcinoma cellline, which expresses a wild-type AR (15). PMCol competition for R1881binding was found to be similar for LNCaP and LAPC4 cells. In addition,the pure anti-androgen bicalutamide was found to have equivalent ARcompetition activity in LNCaP and LAPC4 cells. Therefore, the pureanti-androgen bicalutamide and PMCol were found to possess comparable ARantagonist activity in LNCaP cells, expressing a functional mutant AR,and LAPC4 cells, which express a normal AR.

The AR functions primarily as a transcription factor that is activatedby androgen binding (1). In these studies, the androgen-responsive MMTVpromoter was used to assess modulation of androgen-stimulatedtranscriptional activity. Upon androgen exposure (i.e., R1881), MMTVpromoter activity was stimulated in both LNCaP and LAPC4 cells. Also, inboth cell lines, R1881-stimulation of MMTV activity was significantlyinhibited by PMCol treatment. PMCol treatment alone did not stimulateMMTV promoter activity (i.e., PMCol was not found to have AR agonist orpartial agonist activity). The effects of androgen exposure ontranscriptional activation were further observed by the inhibition ofandrogen-stimulated PSA release after treatment with PMCol in LNCaPcells. Previously, vitamin E succinate was reported to inhibit theeffects of androgen on LNCaP cells through down-regulation of androgenreceptor levels (21). Other agents, such as curcumin, have been shown todecrease AR expression in LNCaP cells (27). In the current study, LNCaPcells treated with 30 μM PMCol for five days did not affect AR proteinlevels. Then, PMCol was found to be a potent inhibitor oftranscriptional activation of androgen-responsive promoters, likelythrough directly blocking AR activation by androgen.

In the current study, PMC, which lacks the phenolic hydroxyl grouppresent on PMCol, was less potent than PMCol at inhibiting androgenicresponses. Therefore, the phenolic hydroxyl group of the chromanol ringcontributes significantly to the anti-androgenic activity of PMCol.Other forms of vitamin E, such as β-, γ-, and δ-tocopherol differ fromα-tocopherol by the number and location of methyl group substitutions onthe chromanol ring (7). The inventors can propose that the antioxidantmoieties of other forms of vitamin E also possess anti-androgenicactivity with potencies that vary dependent on the specific methyl groupsubstitutions present on the chromanol ring.

A variety of dietary agents have been identified that haveanti-androgenic activity in prostate carcinoma cells. However, themechanism of anti-androgenic activity observed by dietary anti-androgensmay vary. For example, curcumin, a component of turmeric, was reportedto down-regulate androgen receptor protein levels in LNCaP cells, whicheffectively attenuates androgenic responses (27). In contrast,indole-3-carbinol, a component of cruciferous vegetables, when convertedto diindolylmethane was reported to act as a potent inhibitor ofandrogen binding in LNCaP cells, but does not affect AR protein levels(28). Zhang et al. (21), have reported that vitamin E succinate isinhibitory to androgenic responses in LNCaP cells throughdown-regulation of AR protein levels, similar to the action of curcumin.By contrast, in the current study, the inventors found that theantioxidant moiety of vitamin E, PMCol, effectively blocks androgenbinding to the AR without affecting AR protein levels, similar toeffects observed with indole-3-carbinol derivatives (28). Therefore,dietary anti-androgens may serve as an effective means of modulatingandrogenic pathways through a variety of mechanisms affecting ARactivity.

PMCol has largely been investigated for its antioxidant activityassociated with being the antioxidant moiety of vitamin E. For example,the antioxidant potency of PMCol was shown to be similar to α-tocopherolin vitro (29). In general, α-tocopherol plasma levels range between 5and 30 μM (30), well within the range of anti-androgenic activityobserved by PMCol in the current study. Due to the high lipophilicity ofvitamin E, it is difficult to assess its anti-androgenic activity bycell culture analysis. However, due to the presence of the highlylipophilic phytyl chain, the subcellular distribution of vitamin E wouldlimit its direct interaction with the AR, which resides in more aqueoussubcellular compartments such as the cytoplasm and nucleus Vitamin E canbe metabolized to derivatives with greater water solubility, such asαCEHC (7, 31), which are structurally similar to PMCol, and may havegreater water solubility and a distinct cellular bioavailabilitycompared to vitamin E. Thus, metabolites of vitamin E may contact the ARin vivo and have anti-androgenic activity, analogous to that produced byPMCol in human prostate carcinoma cells.

In summary, the antioxidant moiety of α-tocopherol, PMCol, was found bythe present inventors to inhibit androgen activity, likely throughcompeting for androgen binding to the AR, with resultant inhibition ofandrogen-sensitive biological pathways. PMCol was not found to possessandrogen agonist or partial agonist activity and hence functions as apure antagonist of androgen activity in the LNCaP and LAPC4 prostatecarcinoma cell lines. Based on the results of the current study, PMColwill serve as a useful agent for modulating androgen activity in vivo.Importantly, the anti-androgenic activity of PMCol poses the possibilitythat the prostate cancer preventive activity of vitamin E may, in part,be due to anti-androgenic effects of vitamin E or metabolites of vitaminE in the prostate. Currently, over 30,000 men die from prostate cancereach year in the United States (4). The prevention of prostate cancerthrough the action of PMCol and derivatives thereof, offers an effectivemeans of reducing the devastation produced by this disease.

Example 2 Acute Oral Toxicity in Mice

The oral toxicity of PMCol was determined in 6 month-old male FVB mice.A single high oral dose of 1000 mg/kg PMCol in sesame oil wasadministered to 4 mice by gavage. Four control mice received only sesameoil by gavage (vehicle control). No significant change in animalbehavior or body mass (FIG. 8A) occurred after administration of PMColor vehicle control for up to 1 week after PMCol administration. In asecond study, four 6 month-old male FVB mice received 200 mg/kg PMColdaily in sesame oil by gavage for 10 days. Three mice receiving onlysesame oil (vehicle control) were used as controls. Body weights weredetermined daily and all mice were autopsied to examine gross organchanges on day 11. No significant difference in body mass change wasobserved in comparing PMCol-treated and vehicle control mice over 10days (FIG. 8B). No gross changes in organs were observed for eitherPMCol-treated or control mice. For example, liver mass was notsignificantly changed in mice receiving PMCol for 10 days (FIG. 8C).Therefore, the LD50 of PMCol in mice is greater than the highest dosetested (i.e., 1000 mg/kg body weight) and PMCol is well tolerated inmice at high doses for up to 10 days.

Example 3

Determining in vivo efficacy of a chroman-derived anti-androgen usingthe LNCaP xenograft model and the TRAMP prostate carcinogenesis model.

A nude mouse/LNCaP xenograft model, similar to the DU145 xenograftmethod previously described (Church et al., Cancer Chemother. Pharmacol.43:198-204 (1999)), may be used to examine the in vivo actions of PMColon human prostate carcinoma cell growth. Male Hsd: athymic nude-nu(nu/nu, BALB/c origin) mice at 4 weeks of age will be acquired fromHarlan Sprague Dawley (Madison, Wis.). At 6 weeks of age, each mousewill be subcutaneously xenografted with 10⁶ LNCaP cells in 0.1 mL ofmedium +0.1 mL of Matrigel (BD Biosciences) in flanking ventral fatpads. One week after LNCaP xenografting, mice will be divided into 5treatment groups of 10 mice each. Mice in group 1 will receive a vehiclecontrol of 0.25 mL of corn oil by gavage, group 2 will receive 25 mg/kgof flutamide (Sigma Chemical Co., St. Louis, Mo.) as an anti-androgentreatment control, group 3 will receive 25 mg/kg of PMCol in 0.25 mLcorn oil, and group 4 will receive 100 mg/kg of PMCol in 0.25 mL cornoil. Dosages are based on toxicity studies described above. Group 5 willbe castrated 1 week after LNCaP xenografting as a low androgen control.Each mouse will be treated daily for 2 months. LNCaP tumor growth willbe determined twice weekly and tumor volume will be determined. Twomonths after LNCaP xenografting, mice will be sacrificed and all LNCaPtumors will be removed and fixed in 10% formalin for histologicalexamination by light microscopy. At the time of euthanasia, blood willbe collected to determine circulating testosterone, luteinizing hormone,and PMCol levels as performed below. Also, livers and male sexualaccessory organs (i.e., seminal vesicles and prostate lobes) will becollected from each mouse for analysis of PMCol's effects on thesetissues.

The TRAMP prostate carcinogenesis model will be used to assess theanti-androgenic activity of PMCol on androgen-dependent tumor growth inthe mouse prostate using a TRAMP mouse colony maintained on a C57BL/6background. At 3-months of age, before the onset of prostatecarcinogenesis, heterozygous male TRAMP mice will be divided into 5treatment groups, as described above. Flutamide's efficacy in the TRAMPmodel has been reported (Raghow et al., Cancer Res. 60: 4093-4097(2000)). TRAMP mice on study will be treated daily. Four months afterthe initiation of treatment, at which point approximately 50% of themice show demonstrable prostatic adenocarcinomas, mice will besacrificed and the prostate lobes and sex accessory glands will beremoved and fixed in 10% formalin and prepared for histologicalanalysis. Hematoxylin and eosin stained slides of prostate glands willbe examined for the presence of prostatic adenocarcinomas, which will bequantified for each treatment group and used to determine the incidenceof prostate carcinomas in control versus treatment groups. Blood willalso be collected to determine circulating testosterone, luteinizinghormone, and PMCol levels as performed below.

Determining the effect of the vitamin E analog PMCol on central nervoussystem feedback control of testosterone and luteinizing hormone bloodlevels compared to PMCol blood levels will be performed as follows.Four-month-old male ICR mice (Harlan Sprague Dawley) will be used toassay the effect of PMCol administration on blood testosterone levels.Mice will be divided into 5 groups of 5 mice each. Mice in group 1 willreceive a vehicle control of 0.25 mL of corn oil by gavage, group 2 willreceive 25 mg/kg of flutamide (Sigma) as a treatment controlantiandrogen, group 3 will receive 25 mg/kg of PMCol in 0.25 mL cornoil, and group 4 will receive 100 mg/kg of PMCol in 0.25 mL corn oil.Group 5 will be castrated as a low androgen control. Mice will betreated daily for 1 month and blood samples will be collected twice aweek by retro-orbital bleed, as previously performed (Church et al.,1999). Blood testosterone levels will be determined using a TestosteroneEIA Test Kit (BioCheck, Inc., Burlingame, Calif.). In addition, thetestosterone blood levels determined by the EIA kit will be validatedusing LC-MS. The luteinizing hormone levels in the blood samples will bedetermined using the Luteinizing Hormone EIA Test Kit (BioCheck, Inc.,Burlingame, Calif.) according to kit instructions. Finally, PMCol bloodlevels will be determined from the samples using LC-MS.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

IV. References

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1. A method for inhibiting the growth of androgen-dependent prostatecancer tumor cells in a human comprising administering to a human havingandrogen-dependent prostate cancer an effective amount of a compoundaccording to the structure:

or a pharmaceutically acceptable salt thereof, which compound bindsandrogen receptors.
 2. A method of delaying the progression ofandrogen-dependent prostate cancer in a human comprising administeringto a human having androgen-dependent prostate cancer an effective amountof a compound having the structure:

or a pharmaceutically acceptable salt thereof, which compound bindsandrogen receptors.
 3. A method of reducing the recurrence ofandrogen-dependent prostate cancer comprising administering to a humanwho has had or presently has androgen-dependent prostate cancer aneffective amount of a compound having the structure:

or a pharmaceutically acceptable salt thereof, which compound bindsandrogen receptors.